The opium poppy, Papaver somniferum L., is an important source of a variety of alkaloids. Due to their narcotic and analgesic properties, opiate alkaloids and their derivates are desired for use in therapy. P. somniferum is a source of clinically useful alkaloids such as morphine, codeine, thebaine, noscapine [also known as narcotine] and papaverine. Morphine is the most abundant opiate alkaloid found in opium and is a powerful analgesic routinely used to reduce pain in humans. Opiate alkaloids are extracted from latex harvested from the green seed pods of opium poppy or from the poppy straw which is the dried mature plant. Morphinan alkaloids are known to derive from the intermediate (R)-reticuline. (R)-reticuline is thought to be formed by its enantiomer (S)-reticuline in a two-step isomerization process. In the first step, (S)-reticuline is transformed to 1,2-dehydroreticuline by the 1,2-dehydroreticuline synthase, and then reduced in the presence of NADPH to (R)-reticuline (FIG. 1). However, genes encoding the enzymes that perform the early conversion steps have not been identified thereby preventing engineering efforts employing the native enzymes.
Cytochrome P450 is a superfamily of monooxygenases with diverse catalytic activities such as oxidation, peroxidation and reduction. The primary chemical reaction catalysed by these monooxygenases is the two electron activation of molecular dioxygen, whereby one oxygen atom is inserted into the substrate with concomitant reduction of the second atom to water. Typically, NAD(P)H provides the required electron equivalents via a number of different redox partners. Many cytochrome P450s have been identified, with one third belonging to the plant kingdom, and found to be involved in the synthesis of a vast number of plant intermediate metabolites such as alkaloids, terpenoids, lipids, glycosides and glucosinolates. Cytochrome P450s are also known to be involved in the metabolism and detoxification of pesticides and other xenobiotic compounds. In eukaryotes, cytochrome P450s are mostly integral membrane bound proteins whereas prokaryotic cytochrome P450s are usually soluble and located in the cytoplasm. Typically, a cytochrome P450 enzyme requires a partnering reductase for the activation of molecular oxygen, which involves transferring two reducing equivalents from NAD(P)H to the heme of the cytochrome P450. Dependent on their redox partner, cytochrome P450 enzymes can be generally divided into two classes. In eukaryotes two component cytochrome P450 systems anchored to the membrane of the endoplasmic reticulum are prevalent comprising a flavin adenine dinucleotide/flavin mononucleotide (FAD/FMN) containing NADPH reductase partnered with a cytochrome P450 flavoprotein. In prokaryotes mainly three component systems can be found comprising an NAD(P)H-binding flavoprotein reductase, a small iron sulphur protein and the cytochrome P450 component. However, naturally occurring fusions of cytochrome P450 systems have also been identified, such as the fusions of the cytochrome P450 heme domain to its flavodoxin redox partner in Rhodococcus rhodocrous strain 11Y, or the soluble monooxygenase cytochrome P450BM3 from Bacillus megaterium comprising a diflavin reductase fused to the cytochrome P450 forming a self-sufficient system. Another self-sufficient fusion has been identified in Rhodococcus sp NCIMB 9784.
Plant cytochrome P450s are known to be involved in alkaloid metabolism and have been successfully cloned, expressed and characterized. For example, WO2009/064771 and WO2008/070274, each disclose cytochrome P450 genes and their use in the alteration of alkaloid content in Nicotiana tabacum. These disclosures describe the inhibition of specific P450s resulting in reduction in the amount of N′ nitrosonornicotine, a known carcinogen, in planta. European application EP11748702.5 discloses cytochrome P450s unique to Papaver somniferum cultivars that produce noscapine. Furthermore, De-Eknamkul W and Zenk M. H. Phytochemistry [1992] Vol 31[3], p 813-821 discloses a 1,2-dehydroreticuline reductase activity that reduces stereospecifically 1,2-dehydroreticuline to (R)-reticuline isolated from Papaver somniferum. The enzyme activity is characterised and shown to be inhibited by (S)- and (R)-reticuline and is only present in morphinan alkaloid producing plants. WO99/35902 discloses mutant Papaver somniferum that show enhanced production of (S)-reticuline although the exact genetic nature of the mutant plants is not fully characterised.
This disclosure relates to a cytochrome P450 and an oxidoreductase from a Papaver somniferum cultivar involved in the two step isomerisation process of (S)-reticuline via 1,2-dehydroreticuline to (R)-reticuline, compounds essential for intermediates in the production of morphinans.